Alexander Drakew

Dentate granule cells in reeler mutants and VLDLR and ApoER2 knockout mice.

We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3-CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that ( 1 ) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.

Developmental distribution of a reeler gene-related antigen in the rat hippocampal formation visualized by CR-50 immunocytochemistry.

During histogenesis of the neocortex, Cajal Retzius cells in the marginal zone express the glycoprotein reelin which is developmentally regulated and involved in the formation of the inside out mode of cortical layering. Cajal Retzius cells are also present in the developing hippocampus. There, inhibition of reelin by blocking with CR-50, an antibody which recognizes the N-terminus of this protein, leads to abnormal development of layer-specific connections. Here we report the developmental distribution pattern of reelin expressing neurons in the rat hippocampal formation using CR-50 immunocytochemistry. Labelled Cajal Retzius cells were located near the hippocampal fissure in neonate rats. Many of these cells were still present in the adult. From postnatal day 4 on, neurons in other layers were stained with the CR-50 antibody. In adult rats immunopositive neurons were found in all hippocampal subfields and in the entorhinal cortex. These observations indicate that in the rat hippocampal formation reelin is expressed in different neuronal types during development and in adulthood. Moreover, Cajal Retzius cells in the marginal zone near the hippocampal fissure are still found in adult animals.

Different primary target cells are important for fiber lamination in the fascia dentata: a lesson from reeler mutant mice.

The factors determining the lamina-specific termination of entorhinal and commissural afferents to the fascia dentata are poorly understood. Recently it was shown that early generated Cajal-Retzius (CR) cells in the outer molecular layer and reelin, synthesized by CR cells, play a role in the lamina-specific termination of entorhinal fibers which form transient synapses with CR cells before establishing their definite contacts with granule cell dendrites (J. A. del Rio et al., 1997, Nature 385, 70-74). By using anterograde tracing with Phaseolus vulgaris leukoagglutinin we show that the normal, sharply delineated entorhinal projection to the outer molecular layer is retained in reeler mutant mice lacking reelin. This coincides with the regular presence of CR cells, the primary, transient target cells of entorhinal fibers. In contrast, the commissural fibers were found to terminate in an abnormal broad, not clearly defined area. This widespread projection coincides with the distribution of granule cells which in the mutant do not form a dense cell layer but are scattered all over the hilus due to a migration defect. Unlike the entorhinal fibers, the commissural fibers arrive in their target layer late in development, when granule cell dendrites are already there. We hypothesize from these results that the presence of the adequate postsynaptic element at the time of fiber ingrowth, CR cells for the early ingrowing entorhinal fibers and granule cells for the late-arriving commissural fibers, is crucial for the normal formation of these layer-specific projections.

Blockade of neuronal activity alters spine maturation of dentate granule cells but not their dendritic arborization

Organotypic co-cultures of the entorhinal cortex and hippocampus were examined to determine the role of the entorhinal fibers in the dendritic development and formation of spines of dentate granule cells. Quantitative analysis of Golgi-impregnated granule cells in single hippocampal cultures and co-cultures with the entorhinal cortex revealed that the presence of entorhinal fibers promoted the elongation and differentiation of the target granule cell dendrites. This was accompanied by an increase in the total number of spines. The contribution of neuronal activity to this afferent-mediated dendritic development was tested by chronic application of the sodium channel blocker tetrodotoxin for 20 days in vitro. Tracing with biocytin showed that the formation of the entorhinohippocampal pathway was unaffected by the blockade of neuronal activity. The dendritic arbor of cultured granule cells and the number of dendritic spines did not differ between tetrodotoxin-treated slices and untreated controls. However, there was a significant increase in the relative number of filiform spines on granule cell dendrites in tetrodotoxin-treated co-cultures. Such filiform spines are a characteristic feature of immature neurons. These results suggest the cooperation of two mechanisms in the dendritic development of dentate granule cells: the specific afferent-mediated dendritic arborization and the activity-dependent maturation of spines.

The hippocampus of the reeler mutant mouse : Fiber segregation in area CA1 depends on the position of the postsynaptic target cells

Area CA1 of the rodent hippocampus is characterized by a highly lamina-specific and nonoverlapping termination of afferent fiber tracts. Entorhinal fibers terminate in stratum lacunosum-moleculare and commissural/associational fibers terminate in strata radiatum and oriens. It has been hypothesized that this fiber lamination depends on specific signals for the different afferent fiber tracts that are located on distinct dendritic segments of the postsynaptic neuron. In order to test this hypothesis, entorhinal and commissural/associational afferents to Ammons horn were investigated in the adult reeler mutant mouse, in which developmental cell migration defects have disrupted the normal array of cells. Golgi staining revealed a deep and a superficial principal cell layer in the mutant. The morphology of the cells located in the deep pyramidal cell layer was considerably abnormal, whereas most cells located in the superficial pyramidal cell layer showed an almost normal cellular and dendritic morphology. Anterograde tracing with Phaseolus vulgaris leukoagglutinin revealed that the duplication and disorganization of the pyramidal cell layer in area CA1 are mirrored by the duplication and disruption of afferent fiber termination zones. In the zone above the abnormal deep pyramidal cell layer, i.e., between the two cell layers, entorhinal fibers as well as commissural/associational fibers terminate and intermingle. In contrast, in the zone above the fairly normal superficial pyramidal cell layer, entorhinal and commissural/associational fibers retain their normal fiber segregation. These data suggest that the normal laminar organization of the murine hippocampus depends on positional cues presented by their target cells.

Stereological estimates of total neuron numbers in the hippocampus of adult reeler mutant mice: Evidence for an increased survival of Cajal-Retzius cells

The cytoarchitecture of the brain is disrupted severely in reeler mice. This is caused by a deficiency in the protein, Reelin, which is essential for the normal migration and positioning of neurons during development. Although cell migration is clearly affected by the reeler mutation, it is believed that the total number of neurons is not. Thus, we were surprised to find an unusually large number of calretinin‐immunopositive cells, presumably Cajal‐Retzius cells, in the molecular layer of the adult reeler hippocampus (Deller et al. [ 1999 ]; Exp. Neurol. 156:239–253). This suggested that the reeler mutation affects the number of neurons in the hippocampus. In order to verify this hypothesis, unbiased stereological methods were employed. Calretinin immunostaining was used as a marker for Cajal‐Retzius cells in control as well as reeler mice and Nissl staining was used to identify hippocampal principal neurons. Total numbers of calretinin‐immunopositive cells, calretinin‐immunoreactive Cajal‐Retzius cells, and Nissl‐stained neurons were estimated in different subfields of the reeler and the control hippocampus. Stereological estimates (P < 0.05) revealed that the total number of calretinin‐immunopositive and Cajal‐Retzius cells in reeler mice are 1.5 and 2.1 times that of controls, respectively. No significant difference in total neuron number was found in any hippocampal subfield. These data demonstrate that the reeler mutation affects the number of calretinin‐immunoreactive Cajal‐Retzius cells in the adult hippocampus, probably due to a reduced excitatory innervation by entorhinal terminals in the absence of reelin. However, the reeler mutation does not affect mechanisms that determine total hippocampal neuron number. J. Comp. Neurol. 439:19–31, 2001.

Abnormal positioning of granule cells alters afferent fiber distribution in the mouse fascia dentata: morphologic evidence from reeler, apolipoprotein E receptor 2-, and very low density lipoprotein receptor knockout mice.

The fascia dentata of the hippocampal formation is characterized by the nonoverlapping and lamina‐specific termination of afferent fibers: entorhinal fibers terminate in the outer molecular layer and commissural/associational fibers terminate in the inner molecular layer. It has been proposed that this fiber lamination depends on the presence of the correct postsynaptic partner at the time of fiber ingrowth during development. Pioneer neurons that guide afferent fibers to their correct layers as well as signals located on granule cells have both been implicated. To study the role of granule cells for the lamina‐specific ingrowth of afferents, the cyto‐ and fiberarchitecture of three mouse mutants (very low density lipoprotein receptor knockout mouse, apolipoprotein E receptor 2 knockout mouse, and reeler mouse) that show different degrees of granule cell migration defects were analyzed. Anterograde tracing with Phaseolus vulgaris‐leucoagglutinin was used to visualize the afferent fiber systems, and immunohistochemistry was used to determine the position of their putative target cells. In controls, granule cells are packed in a single layer. This laminar organization is mildly altered in very low density lipoprotein receptor knockout mice, moderately disturbed in apolipoprotein E receptor 2 knockout mice, and severely disrupted in reeler mice. These changes in granule cell distribution are mirrored by the distribution of commissural fibers. In contrast, changes in granule cell distribution do not severely affect the laminar termination of entorhinal fibers. These data provide further evidence for a role of granule cells in the laminar termination of commissural/associational afferents to the fascia dentata. J. Comp. Neurol. 445:278–292, 2002.

New ways of looking at synapses

Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.

Laminar distribution of synaptopodin in normal and reeler mouse brain depends on the position of spine‐bearing neurons

Synaptopodin is the first member of a novel class of proline‐rich actin‐associated proteins. In brain, it is present in the neck of a subset of mature telencephalic spines and is associated closely with the spine apparatus, a Ca2+ storing organelle within the spine compartment. The characteristic region‐ and lamina‐specific distribution of synaptopodin in rat brain suggested that the distribution pattern of synaptopodin depends on the cytoarchitectonic arrangement of spine‐bearing neurons. To test this hypothesis, synaptopodin was studied in the cortex, striatum, and hippocampus of normal and reeler mice, in which developmental cell migration defects have disrupted the normal array of cells. In situ hybridization histochemistry as well as light‐ and electron microscopic immunocytochemistry were used. In brain of normal mice, the pattern of synaptopodin mRNA‐expressing cells corresponds to that of spine‐bearing neurons and synaptopodin protein is found in a region‐ and lamina‐specific distribution pattern. It is specifically sorted to the spine neck where it is associated closely with the spine apparatus. In brain of reeler mice, the pattern of synaptopodin mRNA‐expressing cells corresponds to that of the abnormally positioned spine‐bearing neurons and the region‐ and lamina‐specific distribution pattern is absent or altered. Nevertheless, synaptopodin was specifically sorted to the spine neck, as in controls. These data demonstrate that the light microscopic distribution pattern of synaptopodin protein depends on the position and orientation of the spine‐bearing neurons. The intracellular sorting process, however, is independent of positional cues. J. Comp. Neurol. 453:33–44, 2002.

Single synapses control mossy cell firing

The function of mossy cells (MCs) in the dentate gyrus has remained elusive. Here we determined the functional impact of single mossy fibre (MF) synapses on MC firing in mouse entorhino-hippocampal slice cultures. We stimulated single MF boutons and recorded Ca2+ transients in the postsynaptic spine and unitary excitatory postsynaptic potentials (EPSPs) at the MC soma. Synaptic responses to single presynaptic stimuli varied strongly between different MF synapses, even if they were located on the same MC dendrite. Synaptic strengths ranged from subthreshold EPSPs to direct postsynaptic action potential (AP) generation. Induction of synaptic plasticity at these individual MF synapses resulted in potentiation or depression depending on the initially encountered synaptic state, indicating that synaptic transmission at MF synapses on MCs is determined by their previous functional history. With these unique functional properties MF-MC synapses control MC firing individually thereby enabling modulation of the dentate network by single granule cells.